生物喋呤合成酶抑制剂对烫伤大鼠金葡菌脓毒症的保护效应及机制

目的　探讨生物喋呤合成限速酶抑制剂 - 2 ,4 二胺 6 羟基嘧啶 (DAHP)对金黄色葡萄球菌 (简称金葡菌 )脓毒症的保护效应及机制。　方法　 5 6只Wistar大鼠随机分为正常对照组、2 0 %TBSAⅢ度烫伤对照组、烫伤后金葡菌感染组和DAHP拮抗组。无菌留取大鼠心、肝、肺、肾组织检测三磷酸鸟苷环水解酶I(GTP CHI)、诱生型一氧化氮合酶 (iNOS)及肿瘤坏死因子α (TNFα )基因表达 ,同时测定组织中四氢生物喋呤 (BH4)和一氧化氮 (NO)的水平。　结果　烫伤后金葡菌感染可导致组织GTP CHI基因表达广泛上调、BH4合成显著增加。同时 ,组织iNOSmRNA表达和NO水平亦明显升高 ,其中肝、肺改变尤为显著。给予DAHP不仅可显著抑制各组织GTP CHImRNA表达和BH4的产生 (P<0 .0 5～ 0 .0 1) ,iNOSmRNA表达和NO的生成亦明显受抑 ,同时TNFα表达也明显降低。此外 ,DAHP拮抗组动物 6h死亡率有所降低 (分别为 2 5 .0 %和 5 5 .6 % ,P =0 .0 8)。　结论　DAHP早期干预可在一定程度上改善革兰阳性菌脓毒症动物的预后 ,其机制可能与DAHP抑制体内BH4和NO的产生有关。     

Significance of biopterin induction in rats with postburn Staphylococcus aureus sepsis

Objective: It has been demonstrated that biopterin, an essential cofactor of nitric oxide synthase (NOS), plays an important role in the pathogenesis of endotoxin-induced shock, yet its biological significance in gram-positive sepsis remains unclear. In this study, we adopted a rat model of postburn Staphylococcus aureus (S.aureus) sepsis to observe the time course and tissue distribution of biopterin in postburn S. aureus infection, and to investigate its potential role in the pathogenesis of gram-positive sepsis. Wistar rats were inflicted with a 20% total body surface area (TBSA) full-chickness scald injury followed by S. aureus challenge, then guanosine triphosphatecyclohydrolase I (GTP-CHI) mRNA expression and biopterin levels in liver, kidneys, lungs and heart were determined at 0. 5, 2, 6, 12 and 24 hours after S. aureus challenge. We found that after S. aureus challenge, GTP-CHI gene expressions and biopterin levels were markedly up-regulated in various tissues, and remained at high values up to 24 hours (P＜ 0. 05-0.01). Meanwhile, the organ function indexes, including serum alanine amimotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), MB isoenzyme of creatine kinase (CK-MB), levels and pulmonary myeloperoxidase (MPO) activities significantly increased at 24 hours postburn, and the multiple organ dysfunction was aggravated by S. aureus challenge. Moreover, it was shown that cardiac GTP-CHI mRNA expression and renal BH4levels were positively correlated with CK-MB and Cr (r=0. 892, P=0. 0012 and r=0. 9423,P=0.0015, respectively). Conclusion: These results suggested that thermal injury combined with S. aureus challenge could induce de novo biosynthesis of biopterin, which acts as the most important cofactor of iNOS, might play a role in the development of multiple organ dysfunction syndrome secondary to postburn sepsis.     

细胞外信号调节激酶在内毒素休克大鼠生物喋呤诱生中的作用机制探讨

目的观察细胞外信号调节激酶（ERK）通路抑制剂对生物喋呤（BH4）和一氧化氮（NO）表达及核因子-kB（NF-kB）活化的影响,探讨内毒素休克时ERK信号通路与NF-kB的交汇作用及其对BH4诱生NO的调控机制。方法采用内毒素休克模型,60只大鼠随机分为正常对照组（n=8）、内毒素休克组（n=32）和ERK抑制剂PD98059拮抗组（n=20）。留取动物肝、肺、肾组织进行NF-kB活性分析以及三磷酸鸟苷环水解酶I（GTP—CHⅠ）、诱生型一氧化氮合酶（iNOS）基因表达的检测,并测定组织及血浆中BH4、NO水平。结果内毒素攻击可导致动物肝、肺、肾组织GTP-CHⅠ基因表达和BH4水平明显升高,至伤后24h仍持续于较高水平;与之相应,组织iNOS基因表达和NO水平亦明显升高;各组织NF-kB迅速活化,并于2h达峰值。采用PD98059处理后,内毒素休克动物肾组织GTP—CHⅠ mRNA表达明显受抑,肝、肺组织GTP—CHⅠmRNA表达仅呈现降低趋势;血浆及肝、肾组织中BH4水平12h显著降低;同样,各组织iNOS mRNA表达及NO水平早期亦显著降低。此外,PD98059处理组动物肝组织2～6h、肺组织2h、24h和肾组织24h时相点NF-KB活性显著降低。结论内毒素休克时抑制ERK通路,能部分下调BH4和NO表达与NF-kB的活化,表明ERK与NF-kB通路间可能存在交汇作用,共同参与了BH4诱生NO的调控作用。     

细胞外信号调节激酶抑制剂对烫伤脓毒症大鼠肿瘤坏死因子-α表达的影响及意义

目的　观察细胞外信号调节激酶 (ERK)抑制剂对烧伤后金黄色葡萄球菌 (金葡菌 )脓毒症动物组织肿瘤坏死因子 (TNF) α表达及多器官功能损害的影响。方法　采用SD大鼠 2 0 %总体表面积Ⅲ度烫伤后金葡菌攻击所致脓毒症模型 ,34只动物随机分为正常对照组 (n =6 )、烫伤对照组 (n=6 )、烫伤后金葡菌感染组 (n =12 )和ERK抑制剂AG12 6拮抗组 (n =10 ) ,检测动物肝、肾、肺组织中ERK磷酸化和TNF α基因 /蛋白表达的改变。结果　烫伤脓毒症后 0 5～ 2 0h肝、肺、肾组织ERK均呈现不同程度的活化 ,其中 2 0h分别为正常对照组的 1 94倍 (P <0 0 5 )、2 86倍 (P <0 0 1)、1 4 1倍。AG12 6拮抗组肺组织磷酸化ERK水平在 2 0h下降 70 6 % (P <0 0 1) ,而肝、肾组织其磷酸化水平不同时相点几乎完全抑制 ,同时各组织中TNF α基因及蛋白表达水平明显下调 (P<0 0 5或 0 0 1)。与烫伤脓毒症组相比 ,AG12 6拮抗组 2 0h肝、肾功能指标明显改善 ,肺组织髓过氧化物酶活性下降 4 0 3% (P <0 0 5 )。结论　ERK信号通路参与了严重烧伤后金葡菌感染所致炎症反应与急性组织损伤的病理过程 ,针对该环节进行早期干预可有效缓解多器官功能异常改变。     

金黄色葡萄球菌肠毒素B单克隆抗体对烫伤脓毒症大鼠急性肺损伤的影响

目的探讨金黄色葡萄球菌(简称金葡菌)肠毒素B(SEB)单克隆抗体(单抗)对烫伤脓毒症大鼠急性肺损伤的保护作用。方法雄性Wistar大鼠56只随机分为正常对照组(n=10)、烫伤对照组(n=10)、烫伤后金葡菌感染组(n=20)和SEB单克隆抗体(单抗)拮抗组(n=16)。测定肺组织SEB水平、髓过氧化物酶(MPO)活性、肿瘤坏死因子(TNF)-α和干扰素(IFN)-γ表达的改变。结果烫伤后金葡菌脓毒症动物肺脏SEB含量明显升高,伤后2、6h分别为66.85ng/g组织和92.46ng/g组织,与正常对照组(14.26ng/g组织)和烫伤对照组(17.32ng/g组织)相比均为P＜0.01;同时,肺组织MPO活性显著增强,峰值可达7.39U/g组织,与正常对照组(2.09U/g组织)相比P＜0.05。与之相应,肺组织MPO活性显著增强(P<0.05)。同时,局部组织IFN-γ和TNF-α基因及其蛋白质表达明显上调(P<0.05),并与肺脏SEB含量呈高度正相关(分别为r=0.9207、P=0.0033和r=0.8142、P=0.0258)。SEB单抗早期干预可有效降低肺组织中SEB含量,并显著抑制IFN-γ和TNF-α的产生,肺脏病理改变亦明显减轻。结论SEB单抗干预可抑制IFN-γ和TNF-α等炎症介质的产生,从而显著减轻烫伤后金葡菌对机体的损害。     

重组杀菌／通透性增加蛋白对内毒素休克中肝脏一氧化氮合酶的 …

目的：探讨重组杀菌／通透性增加蛋白（ｒＢＰＩ２１）对内毒素休克中肝组织一氧化氮合酶（ＮＯＳ）的影响及其意义。方法 大鼠腹腔注射大肠杆菌内毒素（１５．０ｍｇ／ｋｇ）复制内毒素休克模型，动物随机分成正常对照组、内毒素休克组和ｒＢＰＩ２１治疗组。检测肝组织ＮＯＳ活性、三磷酸鸟苷环水解酶Ｉ（ＧＴＰ－ＣＨＩ）活性及生物喋呤含量，同时还观察肝脏微循环血流灌注量的改变。结果 内毒素攻击后肝组织诱生型ＮＯＳ（ｉＮ     

烫伤合并金葡菌感染大鼠组织CD14 mRNA的改变

目的　探讨细菌脂多糖受体CD14在烫伤合并金黄色葡萄球菌 (金葡菌 )感染中的变化规律及其意义。　方法　采用大鼠 2 0 %总体表面积Ⅲ度烫伤合并金葡菌攻击造成脓毒症模型 ,动态检测心、肝、肺、肾等重要器官中CD14mRNA表达的改变 ,同时观察内毒素在动物循环及主要脏器内的分布特点。　结果　烫伤合并金葡菌脓毒症早期 ,各脏器内毒素含量即明显高于正常对照组 ,并于2～ 6h达峰值 ,其中以肝、肺组织内毒素水平升高幅度最为显著 (P <0 .0 5 )。而血浆内毒素水平亦于伤后 2h显著高于正常对照组 (分别为 0 .30 5 6EU/ml和 0 .12 5 0EU/ml,P <0 .0 5 )。与此同时 ,小肠组织中二胺氧化酶的活性明显降低 (P <0 .0 5 )。烫伤合并金葡菌感染后 ,各组织CD14mRNA的表达亦呈不同程度升高 (P <0 .0 5 ) ,其中肺脏改变尤为显著 ,伤后 6、2 4h肺脏CD14mRNA表达分别为正常对照组的 1.80和 1.81倍。　结论　烫伤合并金葡菌攻击可导致内毒素移位和组织CD14mR NA表达不同程度升高 ,CD14基因表达的上调可能与移位内毒素的刺激作用有关。     

脓毒症大鼠生物喋呤的组织分布特点和意义

目的　探讨腹腔感染致脓毒症时重要器官生物喋呤及其合成限速酶基因表达的改变和病理生理意义。方法　腹腔感染致脓毒症模型采用盲肠结扎穿孔法(CLP),用反相高效液相分析法和逆转录-聚合酶链反应方法测定24只大鼠肝、肺、肾等组织生物喋呤含量及三磷酸鸟苷环水解酶I(GTP-CHI)mRNA的表达。结果　脓毒症大鼠2h时肝、肺、肾组织生物喋呤含量显著增多［分别为(4.18±0.16)、(2.71±0.32)、(2.45±0.27)ng/g蛋白］,同时不同组织GTP-CHImRNA表达亦明显增强,各脏器功能均表现不同程度地损害(P＜0.05)。相关分析显示,肝、肺组织生物喋呤与反映相应脏器功能的指标呈高度正相关(分别为r=0.7916,P＜0.001和r=0.8004,P＜0.001)。结论　生物喋呤参与了腹腔感染所致脓毒症的发生、发展过程。     

金葡菌肠毒素B在烧伤脓毒症大鼠中的分布特点及意义

目的　探讨金葡菌肠毒素在烧伤后G 菌脓毒症发生、发展过程中的意义。　方法　采用大鼠 2 0 %TBSAⅢ度烫伤复合金葡菌攻击造成脓毒症模型 ,观察金葡菌肠毒素B(SEB)在动物血浆及心、肝、肺、肾等重要器官中的分布 ,同时动态检测相关器官功能指标的改变。　结果　烫伤脓毒症早期 ,动物血浆中SEB含量呈一过性升高 (P <0 .0 1) ,继而逐渐下降。心、肝、肺、肾等组织中SEB含量于金葡菌攻击后 2h即明显升高 (P <0 .0 1) ,且持续上升。烫伤后 2 4h动物多器官功能明显受损 ,金葡菌的攻击可进一步加重多脏器功能损害 ,且肝、肾等器官损害程度与组织SEB含量呈显著正相关 (P <0 .0 5～ 0 .0 1)。　结论　金葡菌肠毒素在烫伤脓毒症诱发的多器官功能损害中具有一定作用     

己酮可可碱对内毒素休克家兔生物喋呤诱生的干预作用

目的　观察己酮可可碱 (PTX)对内毒素休克家兔休克时生物喋呤诱生的影响。　方法　 2 8只大耳白兔 ,随机分为 3组 ,对照组 (8只 )、内毒素休克组 (10只 )、PTX治疗组 (10只 )。制作内毒素休克模型 ,观察血浆生物喋呤、肿瘤坏死因子及组织三磷酸鸟苷环水解酶I活性的变化 ,同时监测全身血流动力学改变。　结果　内毒素休克大鼠早期给予PTX治疗 ,可显著降低血浆生物喋呤及肿瘤坏死因子水平 ,与对照组及休克组比较 ,差异有显著性意义 (P <0 0 5 ,P <0 0 1) ;不同程度地抑制肝、肺、心等组织三磷酸鸟苷环水解酶I活性 (P <0 0 5 ) ;与此同时 ,治疗组平均动脉压、心输出量及外周血管阻力均比休克组明显提高 ,差异有显著性意义 (P <0 0 5 ,P <0 0 1)。　结论　PTX能有效抑制内毒素休克时体内生物喋呤的合成与释放 ,明显减轻全身血流动力学障碍。     

内毒素休克大鼠核因子κB活化规律及其在生物蝶呤诱生中的作用

目的观察内毒素休克大鼠血浆及主要脏器核因子(NF)κB活化规律及其对生物蝶呤(BH4)和一氧化氮(NO)表达水平的影响,探讨内毒素休克时NF-κB信号通路对BH4诱生NO的分子调控机制及其与多器官功能损害的关系。方法将47只大鼠按表格随机法分为正常组(8只)、内毒素／脂多糖(LPS)组(24只,每观察时相点8只,均同时注射LPS制成休克模型)和拮抗组[15只,每观察时相点5只,均同时注射LPS并以吡咯烷二硫代氨基甲酸盐(PDTC)拮抗]。休克及拮抗组于注射LPS后2、6、12 h观察,并与正常组同法处死,无菌留取大鼠血标本及肝、肺、肾组织,测定组织中NF-κB活性和三磷酸鸟苷环水解酶Ⅰ(GTP-CHⅠ)和诱导型一氧化氮合酶(iNOS)mRNA表达水平、血浆和组织中的BH4含量及NO水平、肝脏和肾脏功能指标、肺组织髓过氧化物酶活性。结果与正常组(例如肺组织中NF-κB活性为26±6)比较,LPS组大鼠组织中NF-κB迅速活化(P<0．01),并于注射后2 h达峰值(肺组织中为291±44);LPS组各组织中GTP-CHⅠ和iNOS mRNA表达、BH4和NO水平也较正常组明显升高(P<0．05或0．01),至伤后12 h仍持续较高水平。此外,该组相应器官功能均受到不同程度的损害。应用PDTC的拮抗组大鼠各组织中NF-κB活性均较LPS组有所降低,GTP-CHⅠ、iNOS mRNA表达及BH4、NO水平显著受抑,肝、肺、肾功能明显改善。结论内毒素休克时机体内NF-κB通路高度活化,并对BH4／NO系统具有明显调节效应;可通过下调BH4介导的iNOS的过度活化抑制NF-κB信号途径,从而减轻组织炎性反应,对机体脏器功能起到保护作用。     

The effect of indomethacin on burn-induced immunosuppression

Recent interest in the role of prostaglandin inhibitors as immunomodulators following major injury prompted us to study the effect of indomethacin on burn-induced immunosuppression in rats as measured by the delayed-type hypersensitivity (DTH) skin test response, ability to contain an intradermal bacterial challenge (10(8) Staphylococcus aureus 502A injected intradermally), and overall survival from spontaneous burn wound sepsis. Fifty male Sprague-Dawley rats sensitized to keyhole limpet hemocyanin (KLH) were subjected to a 30% full-thickness scald burn. Group 1 (n = 24) received indomethacin at 0.5 mg/kg intraperitoneally once daily with the first dose given immediately following the burn. Group 2 (n = 24) received vehicle only. Prostaglandin E2 measured by radioimmunoassay on day 17 was 2553 +/- 832 pcg/ml serum (+/- SEM) in the vehicle group and 1042 +/- 231 pcg/ml in the indomethacin group (P = 0.058, unpaired t test). Burn injury induced a decrease in the DTH response to KLH and an increase in the Staph lesion size (P less than 0.05) which was not corrected by indomethacin treatment. All animals developed spontaneous burn wound sepsis by day 14. Survival after 17 days in the indomethacin group was 100% compared to that of the vehicle group, 79%, P less than 0.05 (Fisher exact test). We conclude that despite unmeasurable corrections of the burn-induced suppression of the DTH response and local nonspecific bacterial defenses, low-dose indomethacin improves survival following burn sepsis.     

氯化镧对内毒素/脂多糖诱导巨噬细胞株一氧化氮合酶表达的抑制作用

目的了解氯化镧（LaCl3）对内毒素／脂多糖（LPS）刺激的巨噬细胞诱导型一氧化氮合酶（iNOS）表达的影响，并探讨其机制。方法将小鼠巨噬细胞株RAW264．7分为空白对照组、LaCl、组、LPS组和LaCl3＋LPS组。前3组细胞分别用常规培养液、含2．50μmol／L LaCl3的培养液、含1mg／L LPS的培养液培养24h，LaCl3＋LPS组用含2．5μmol／LLaCl，的培养液培养24h后，换为含1mg／L LPS的培养液培养24h。采用免疫细胞化学染色法检测iNOS在各组细胞中的表达强度；蛋白质印迹法检测iNOS的蛋白表达水平；反转录一PCR测定iNOS的mRNA表达水平；硝酸还原酶法测定各组细胞培养上清液中一氧化氮（NO）含量。结果免疫细胞化学染色结果显示，iNOS主要分布于各组细胞的胞质中，空白对照组和LaCl3组荧光强度极弱；LPS组荧光强度最强，阳性细胞百分率为44．4％，明显高于LaCl3＋LPS组（11．8％，P〈0．05）。LPS组iNOS蛋白及其mRNA表达量和细胞培养上清液中NO含量均高于其余各组（P〈0．05）。结论LaCl3可在mRNA水平和蛋白水平抑制LPS诱导的iNOS过度表达，减少NO生成，提示LaCl3能拮抗LPS诱导的iNOS过度活化。     

胰岛素干预对严重烧伤后早期大鼠血管内皮细胞的保护效应

目的了解胰岛素对严重烧伤后早期大鼠血管内皮细胞的保护效应，并分析相关机制。方法将sD大鼠分成假伤组（7只）、烧伤组（7只）和处理组（7只）。后2组制成30％TBSAⅢ度烧伤（用94℃水浴烫伤）模型，假伤组37℃水浴模拟致伤过程。伤后即刻，各组经腹腔注射等渗盐水（40ml／kg）抗休克，同时处理组皮下注射胰岛素3U／kg、另2组同法注射等体积等渗盐水。伤后24h透射电镜下观察各组大鼠主动脉内皮细胞形态及结构，检测其血糖、血清一氧化氮（NO）及各型一氧化氮合酶（NOS）水平。结果镜下可见，处理组较烧伤组主动脉内皮细胞受损程度明显减轻。伤后24h，假伤组、烧伤组、处理组大鼠血糖分别为（4．9±0.8）、（8．2±1.0）、（7．1±0.．7）mmol/L，后2组均显著高于假伤组（P〈0．01），但处理组明显低于烧伤组（P〈0．05）。处理组血清NO、总NOS和结构型NOS（cNOS）水平均明显高于烧伤组（P〈0．01），但2组血清诱导型NOS水平比较，差异无统计学意义（P〉0、05）。结论胰岛素干预可保护严重烧伤后早期大鼠的血管内皮细胞，其机制可能与促cNOS水平升高从而使生理态NO合成增加有关。     

Nitric oxide modulates fracture healing.

The role of the messenger molecule nitric oxide has not been evaluated in fracture healing. NO is synthesized by three kinds of nitric oxide synthase (NOS): inducible NOS (iNOS), endothelial (eNOS), and neuronal (bNOS). We evaluated the role of these enzymes in a rat femur fracture-healing model. There was no messenger RNA (mRNA) expression, immunoreactivity, or enzymatic activity for NOS in unfractured femoral cortex. After fracture, however, mRNA, protein, and enzymatic activity for iNOS were identified in the healing rat femoral fracture callus, with maximum activity on day 15. The mRNA expression for eNOS and bNOS was induced slightly later than for iNOS, consistent with a temporal increase in calcium-dependent NOS activity that gradually increased up to day 30. mRNA expression for the three NOS isoforms also was found in six of six human fracture callus samples. To study the effect of suppression of NO synthesis on fracture healing, an experimental group of rats was fed an NOS inhibitor, L-nitroso-arginine methyl ester (L-NAME), and the control group was fed its inactive enantiomer, D-nitroso-arginine methyl ester (D-NAME). An 18% (p < or = 0.01) decrease in cross-sectional area and a 45% (p < or = 0.05) decrease in failure load were observed in the NOS-inhibited group on day 24 after fracture. Furthermore, the effect of NO supplementation to fracture healing was studied by delivering NO to the fracture site using carboxybutyl chitosan NONOate locally. On day 17 after fracture, there was a 30% (p < or = 0.05) increase in cross-sectional area in the NO-donor group compared with the NOS inhibition group. These results show for the first time that NO is expressed during fracture healing in rats and in humans, that suppression of NOS impairs fracture healing, and that supplementation of NO can reverse the inhibition of healing produced by NOS inhibitors.     

Reperfusion liver injury induces down-regulation of eNOS and up-regulation of iNOS in lung tissues

OBJECTIVES: Acute lung injury and inflammation can occur after hepatic ischemia/reperfusion (I/R). Little is known regarding the possible role of nitric oxide synthase expression in this complex type of lung injury. METHODS: Real-time polymerase chain reactions and immunohistochemistry were used to assess the mRNA and protein expression of eNOS and iNOS in lung tissue after I/R challenge to the liver. Ischemia was induced by clamping the hepatic artery and portal vein for 40 minutes. After flow was restored, the liver was reperfused for 300 minutes. Blood samples were collected to assay three inflammatory parameters: tumor necrosis factor (TNF)-alpha, hydroxyl radicals, and NO. Lung lavage samples were assayed for protein and myeloperoxidase. The expression of eNOS and iNOS in lung tissues (n = 3) was also evaluated after I/R challenge to the liver. The iNOS inhibitor aminoguanidine was also tested in this I/R model. RESULTS: Reperfusion of the liver produced increased blood concentrations of TNF, hydroxyl radicals, and NO (P < .001; n = 8). Bronchial lavage fluids showed higher levels of protein and myeloperoxidase in the I/R than in the sham-treated group (P < .01). eNOS expression was down-regulated and iNOS expression up-regulated in I/R lung tissues (n = 3). The iNOS inhibitor aminoguanidine (10 mg/kg) significantly attenuated the lung injury. CONCLUSIONS: I/R injury to the liver induced lung injury involving systemic inflammatory responses and iNOS expression. Administration of aminoguanidine significantly attenuated the injury, suggesting that iNOS expression may play a critical role in lung injury induced by I/R of the liver.     

一氧化氮合成酶在大鼠小肠移植急性排斥反应中的作用

目的 观察神经型(nNOS)和诱导型一氧化氮合成酶(iNOS)在大鼠小肠移植急性排斥反应(AR)中作用.方法 行大鼠原位小肠移植.实验分为2组.1组:同系移植组(Lewis→Lewis,12例);2组:同种移植组(DA→Lewis,12例).观察术后生存时间.再灌注30 min、术后1、3、5、7d检测血清一氧化氮(NO)浓度;开腹行麦芽糖吸收实验;切取移植肠管,苏木素-伊红(HE)染色后光镜检查.免疫组织化学法观察移植肠nNOS和iNOS的活性.逆转录-聚合酶链反应(RT-PCR)法检测移植肠nNOS mRNA和iNOS mRNA的表达.结果 A组生存时间＞30 d.B组生存时间为(6.83±0.75)d.再灌注后A组nNOS染色与mRNA表达明显减弱,此后nNOS染色和mRNA表达分别于术后3、7d恢复正常.再灌注后A组iNOS染色与mRNA表达增强,此后逐渐减弱.与A组比较,术后3～7 d,B组nNOS染色减弱,iNOS染色增强,血清NO水平明显升高(P＜0.05),血糖吸收值显著降低(P＜0.01);术后5、7d,B组nNOS mRNA表达显著下降(P＜0.001),iNOS mRNA表达明显增强(P＜0.01).结论 在AR过程中,nNOS可能调节了iNOS的表达;nNOS的活性和表达与移植肠管的结构和吸收功能密切相关;iNOS的激活是加重组织损伤的重要因素之一.     

Dual effects of nitric oxide in acute colon obstruction

Nitric oxide (NO) plays central role in the pathophysiology of large bowel diseases. In the gastrointestinal tract the predominant form of nitric oxide synthase (NOS) isoenzymes is neuronal NOS (nNOS). The aims were to investigate the role of NO and the activation of NOS isoforms during acute colonic obstruction. Haemodynamic changes, large bowel motility and plasma levels of nitrate-nitrite (NOx) were observed for 7 hrs in anaesthetized dogs. Group 1 (n=6) served as sham-operated control. In groups 2 (n=8), 3 (n=6), and 4 (n=6) colon obstruction was initiated. Groups 3 and 4 were treated with non-selective NOS inhibitor N-nitro-L-arginine (NNA, 4 mg/kg) or with the selective nNOS inhibitor 7-nitroindazol (7-NI, 5 mg/kg) 3 hr after the obstruction. At the end of the experiments, tissue biopsies were taken from the oral and aboral parts of the colon to determine the constitutive and inducible NOS (cNOS and iNOS, respectively) activities. RESULTS: The cNOS activity of the colon was significantly higher orally then aborally in each group. After obstruction the characteristic features of hyperdynamic sepsis were observed. The obstruction caused significant increase in iNOS activity, which was significantly reduced by the NOS inhibitors. The obstruction increased the motility on both parts of the colon. The administration of NNA transiently inhibited, but later significantly increased the motility of the colon segments. Inhibition of nNOS by 7-NI treatment did not influence the hemodynamic parameters but decreased the motility. CONCLUSION: Neuronal NO increases colon motility at the early stage of large bowel obstruction, however, during a concomitant sepsis the excess of inducible NO will moderate this effect.     

Studies on B-lymphocyte dysfunctions in severely burned patients

We studied in vitro functional parameters of peripheral blood B-lymphocytes from severely burned patients (n = 10; burn injuries ranging from 25 to 72% TBSA). While the number of B-cells remained unchanged, B-cell proliferation induced by Staphylococcus aureus strain Cowan I (SAC) was normal or even enhanced at early and late phases postburn, but showed a marked suppression during the second to fourth week. A similar pattern was observed for the pokeweed mitogen (PWM)- or SAC-stimulated synthesis of immunoglobulin M (IgM), whereas IgG production was decreased over the whole postburn period monitored. Cytokine (interleukin 4)-induced B-cell activation as indicated by the expression of the CD23 surface antigen was impaired throughout the second to fifth week. In parallel, the release of the proteolytic cleavage product sCD23 which represents a B-cell growth and differentiation factor was reduced. Our data provide evidence that activation, proliferation, and differentiation processes of B-lymphocytes are impaired in severely burned patients, which may contribute to their enhanced susceptibility to infection and sepsis.